The insulin-like growth factors (IGFs) constitute a family of proteins having insulin-like and growth stimulating properties. The peptides have been purified from plasma, and their structure and chemical properties have been determined. The IGFs show close structural homology with proinsulin and elicit similar biological effects (Rinderknecht,E. and Humbel, R. E.: J. Biol. Chem., 253, 2769-2773 (1978), Zapf,J. et al.: Curr. Topics in Cell Reg., 19,257-309 (1981), Humbel,R. E.: Hormonal Proteins and Peptides, 12, AP 56-69 (1984), van Wyk,J. J.: Hormonal Proteins and Peptides, 12, AP 81-125 (1984)). Although being members of the same family of proteins, the two main forms of IGFs are rather different with respect to their chemical characteristics. IGF-1 is a basic peptide (pI 8.4) and shows a 43% homology with proinsulin (Rinderknecht,E. and Humbel,R. E.: J. Biol. Chem., 253, 2769-76 (1978). IGF-2 is an almost neutral peptide (pI 6.4) shows a 60% homology with IGF-1.
IGF-1 consists of 70 amino acids, the sequence of which has been determined by Rinderknecht,E. and Humbel,R. E.: J. Biol. Chem, 253, 2769-73 (1978). The physical-chemical parameters for IGF-1 have been studied extensively by several groups. The material used for these studies was primarily isolated from human plasma by a variety of conventional chromatographic methods. These include initial plasma fractionation and further precipitation of other proteins using acid ethanol extraction to remove binding proteins and other plasma proteins. The acidified IGF-1-enriched fraction was then subjected to cation exchange chromatography, and several steps of reversed phase and isoelectric focusing resulting in the collection of a few .mu.g of purified IGF-1.
IGF-1 and to a lesser degree IGF-2 are found in plasma, but only a minor fraction is present in a free form. Specific binding proteins of high molecular weight having very high binding capacity for both IGFs act as carrier proteins or as modulators of IGF functions (Holly,J. M. P. and Wass,J. A. H.: J. Endocrinol, 122,611-618 (1989), Ooi,G. T., and Herrington,A. C.: J. Endocrinol., 118, 7-18 (1988)). The IGFs exert an insulin-like effect when present in high amounts although only to an extent of 1% of that of insulin. The somatic function of IGF-1 is formulated in the so-called "Somatomedin Hypothesis", suggesting that growth hormone released from the pituitary gland exerts its effect by stimulating IGF-1 release from the liver, which IGF-1 then mediates the somatogenic actions via binding to cell surface receptors in the target tissues (Salmon,W. D. Jr., and Daughaday,W. H.: J. Lab. Clin. Med., 49, 825-836, (1957). The principal local biological function of IGFs is as a potent mitogenic agent, although the effect is weaker than that measured e.g. for PDGF and FGF (Froesch et al.: Ann. Rev. Phyiol. 47, 443-467 (1985). Together with PDGF, IGF-1 shows synergistic biological effects (Kato et al.: Eur. J. Biochem., 129, 685-690 (1983), Stiles et al.: P.N.A.S., USA 76, 1279-1283 (1979)). The principal biological effects of IGF-1 are thus considered the function of propagating mitogenic responses, and of maintaining growth initiated by other factors under properly controlled conditions (O Keefe et al.: Mol. Cell. Endocrinol., 31, 167-186 (1983) .
The potency of IGF-1 as a mitogenic factor and the vast potential use thereof in e.g. wound healing and in nitrogen metabolism has encouraged a great number of biopharmaceutical companies to try to express IGF-1 in various organisms due to the small amount present in human plasma. Production of IGF-1 in yeast is favored by a very easy purification of a correctly folded polypeptide. However, there are certain significant drawbacks associated with the use of these expression systems. The yield of fermentation is low (few mg per liter of fermentation broth), and the risk of obtaining O-linked glycosylated forms of the molecule is significant (Gillerfors et al.: J. Biol. Chem., 264, 2748-53), (1989).
Expression in bacteria has until now been the most successful approach giving high yields of IGF-1 and IGF-2, but mostly in cell associated forms. It has also been tried to express IGF-1 fused to a stabilizing peptide. Such a peptide is normally of the same size as IGF-1 itself, and may have physico-chemical properties facilitating the purification of the resulting protein. The fusion protein is cleaved by either chemical or enzymatic cleavage, leaving the mature protein for isolation.
Extraction of the recombinant peptide (fusion peptide) is normally associated with a denaturating step followed by in vitro renaturation of the extracted IGF-1, resulting in the native conformation of IGF-1.
In the bacterial systems described so far, yields of from 1 to 1.5 grams per liter of fermentation broth have been described for biosynthetic expression of hGH. However, despite the high yield of expression only minor amounts of native IGF-1 is obtained because of uncontrolled formation of intra and inter molecular disulphide bridges. Therefore, bacterial expression of products containing such disulphide bridges has to be followed by in vitro renaturation during the later purification of the polypeptides which normally results in extensive losses of product.
The object of the present invention is primarily to overcome the problems described in the prior art with respect to producing IGF-1 in microorganisms in high yields.
As used in the present context, the expression "folding" is used to designate the process of establishing the tertiary structure of the protein, including the formation of the intramolecular disulphide bonds.
The expression "Renaturation" is used to designate the establishing of the native tertiary structure of the protein.
The expression "Denaturing" is used to designate the break-down of the tertiary structure of the molecules disrupting the disulphide bonds and/or uncoiling of the protein.